The production of heterologous proteins in plants for various purposes is a fast-growing field of study. Plants as biofactories for the production of proteins is a new technology that is being employed by a number of groups for edible vaccines, pharmaceuticals and industrial enzymes. Hood, E. and Howard, J. Protein Products from Heterologous Plants. Agro-Food-Industry Hi-Tech, 3, Vol. 10, May/June 1999, pp. 35-36 Hood, E. and Jilka, J. (1999) Plant Based Production of Xenogenic Proteins. Current Opinion in Biotechnology, 10:4, pp. 382-386. Pharmaceutical and vaccine production in plants has several advantages in that the material contains no contaminating organisms and can be directly consumed. Production of industrial enzymes in plants provides the possibility of considerably reduced production costs, the benefit of recovered costs through sale of by products, easier transportation and reduced chance of contamination.
Over-expression of a protein in a heterologous plant requires quite high expression levels to make the system economically viable, a condition that has been achieved for a number of proteins, e.g. the diagnostic protein, avidin (U.S. Pat. No. 5,767,379); aprotinin (U.S. Pat. No. 5,824,870); hepatitis vaccine (U.S. Pat. No. 5,914,123); Transmissible Gastroenteritis vaccine (U.S. Pat. No. 6,034,298); viral vaccines (U.S. Pat. No. 6,136,320); proteases (U.S. Pat. No. 6,087,558) and laccase (WO 00/20615). Using plants as biofactories for pharmaceutical and industrial enzyme production provides considerable advantages over traditional methods of such protein production, since plants provide easier transport and cost savings, but also can be far more readily produced in large quantities than when produced in bacteria or fungi for example, allowing for even further increases in the amount of enzyme which may be produced.
Achieving high levels of enzyme production in plants is impacted by several factors, such as location of expression of the protein within specific tissues and within particular subcellular compartments to insulate the plant tissues from the activity of the protein. Thus, in WO 00/20615, it is discussed that preferentially directing expression to the seed of the plant and also to plant cell wall tissue and to the endoplasmic reticulum of the plant cell is advantageous in increasing protein production. Attempts continue to yet further increase expression levels of the heterologous proteins in plants to provide optimum efficiency, efficacy and decrease costs.
When choosing a variety of plant in which to introduce a heterologous nucleotide sequence in order to express a heterologous protein, two approaches have been typical. One is to select those varieties or lines that have good agronomic traits. These so-called “elite” plants primarily demonstrate high yield. They also may have traits that make them better able to withstand adverse weather conditions in the area in which they are grown, or withstand disease or insect attack better than other plants. If the output of grain can be increased, it is believed, the amount of heterologous protein produced will be increased. Indeed, this can be a successful approach. As a result, there is little incentive to select non-elite plants that demonstrate poor agronomic traits.
Another approach which has been used in selecting plants for heterologous protein production is to choose a plant which has a protein sink, that is, where one or more of the plant proteins is reduced as compared with the naturally occurring wild-type version of the variety or line. In this approach, a promoter, which directs the heterologous protein expression to the area of the sink, or protein depletion, is expected to provide for increased expression levels of the heterologous protein. For example, in one study, Takaiwa used rice plants with reduced glutelin levels in the endosperm to express heterologous protein. This rice had reduced glutelin levels, and is not a rice with reduced levels of alcohol soluble proteins. A nucleotide sequence expressing the desired protein was linked to an endosperm promoter and introduced into the plants. Takaiwa, F. “Development of high accumulation systems in rice endosperm” Abstract, New Frontier of Plant Molecular Farming; NIAR, Tsukuba City Japan, Mar. 7-8, 2000. The result, as expected, was increased expression levels of the heterologous protein in the endosperm.
The inventors have surprisingly found that one can obtain more plants with higher expression levels of heterologous protein by selecting host plants in a manner contrary to what is known about plant expression systems. They have found that if a plant is selected which has reduced alcohol-soluble protein levels in the endosperm, significantly higher expression levels of heterologous protein are achieved in the embryo. Expression levels of two to three times that in plants which do not have reduced alcohol soluble levels are obtained. Thus, a sink is created in one part of the plant tissue, but protein levels actually increase elsewhere. Impacting the endosperm causes increased levels of heterologous protein accumulation in the embryo.
Normally, plants with reduced alcohol-soluble protein levels, the opaque mutants for example, have decreased protein levels in the endosperm and the embryo has increased levels of saline-soluble water-insoluble proteins, such as globulins. Puckett, J. L. and Kriz, A. L. “Globulin Gene Expression in Opaque-2 and Floury-2 Mutant Maize Embryos” Maydica (1991) 36:161-167. (see p. 162). However, the inventors have found that the amount of heterologous protein is increased considerably in such plants. It is especially surprising when heterologous water-soluble proteins are introduced into the plant, the levels of such heterologous protein production are increased, even though there has been an increase in the embryo of native water-insoluble proteins. Not only is the sink “filled” elsewhere, but it is filled with a non-native protein, and can be filled with a non-native protein that is quite different from that which is depleted.
The inventors have discovered that by introducing nucleotide sequences encoding heterologous protein into plants that have reduced levels of alcohol soluble proteins in the endosperm, there is an increase in the expression level of the heterologous protein. This is unexpected for several reasons. First, the literature indicates protein levels decrease in such plants. Second, if the sink created was to be filled, one would expect native plant protein to fill the sink, not heterologous protein. Third, the sink is created by reduction of alcohol-soluble proteins in the endosperm but the heterologous protein is increased as measured in nanograms of protein per milligram of dry weight of plant seed. Also, the zein content of the seed is only about 8% of the seed weight. In plants having reduced zein content, the amount can be decreased by 30% to 90%. However, the increases obtained in heterologous expression are two to three times that of expression in a plant not having reduced alcohol soluble proteins in the endosperm. Finally, the levels of heterologous soluble protein expression are particularly high in the embryo of the seed of the opaque plant, which is surprising given that water insoluble proteins increase in the embryo as noted by Puckett, supra.
Without wishing to be bound by any theory, it is believed that when there are reduced proteins in the endosperm, somehow the plant “responds” to the heterologous protein as a globulin-like protein and fills the embryo, even to the exclusion of native globulins, and even though the heterologous protein is water-soluble, not water-insoluble, as globulins are. Ranges of expression are recovered, and the levels of expression are overall higher using these plant backgrounds.
The inventors have found that plants with reduced levels of alcohol soluble protein levels in the endosperm provide a good host in which to express heterologous proteins. It is particularly surprising to use this type of plant, since it typically shows such poor agronomic traits. For example, the opaque mutants have been studied in the past as a potential source of germplasm to increase lysine content and nutrition in corn, but were found to have low yield and susceptibility to disease. Seed of the plant exhibit a soft, chalky, non-transparent appearance, with very little hard vitreous or horny endosperm. Hence, the name opaque was applied to such mutants. Because of these characteristics, they are more prone to damage by seed rot, insects, rodents and harvesting damage. In fact, it has been stated that “[d]ue to the reduction in seed weight and total protein content, the double mutant has no practical interest in breeding maize for quality.” Salamini, et al, “Mucronate, Mc, a dominant gene of maize which interacts with opaque-2 to suppress zein synthesis” Theor. Appl. Genet. (1983) 65:123-128. Here, however, the inventors have found an advantage in selecting for such plants as a source of heterologous protein.
In addition, the inventors have also found that high oil plants are a desirable choice of plant host for expressing heterologous protein.
High-oil content plants have been studied for some time for their improved nutritional value as animal grain. For example, maize is an important cereal crop used for livestock feeding, commercial products and human consumption. Increasing oil content adds value to such products. Much of the work involving high-oil plants has been devoted to increasing yield while retaining the high-oil content trait. See e.g., Alexander, Maydica 44 (1999)222-112. High-oil hybrids with greater than 6% by dry weight oil content are lower in yield than hybrids with lower levels of oil. These plants, such as corn, have been transformed with heterologous nucleotide sequences, such as in Asrar, U.S. Pat. No. 6,091,002 in which polyhydroxyalkanoate (PHA) polymers are produced in high oil plants since these plants produce carbon substrates which can be employed for PHA biosynthesis. Reports on whether there is increased protein content correlated with high oil phenotype have varied. See, for example, Kovacs-Schneider et al. Novenytermeles 35 (1986): 383-389 in which they discuss selection for high oil content in corn correlated with a simultaneous increase in protein content attributed to an increase in embryo size. In soybeans, it has been noted that protein increases insignificantly in high oil types. Qiu, L. et al., Scientia Agricultura Sinica (1990) 23, No. 5: 28-32. In cotton, there is a negative correlation of protein content with seed oil. Zhou, Z. G., et al. Shaanxi Journal of Ag. Sci. (1992) 3: 3-5. Thus statements regarding protein content in high oil plants have been highly contradictory.
Regardless of whether or not high oil plants are correlated with high protein, the inventors have found that not all plants with higher protein content are acceptable hosts for production of heterologous protein. The Illinois High Protein variety, for example, is, as its name implies, a hybrid with increased protein levels in the seed. However, the inventors have found that it is not a good host, and that heterologous protein levels are quite low using this plant. However, if high oil plants are the host for heterologous protein expression, heterologous protein levels are significantly increased. Further, the heterologous protein is apparently out-competing the native protein already present in the embryo. High oil plants do not have a “sink” as with the low alcohol soluble protein plants discussed above. In this situation, the heterologous protein increases rather than native protein acting to limit heterologous protein expression.
In addition, the inventors have found that when the heterologous protein is introduced into a cross combining the high oil and opaque plants, yet further increases in heterologous protein expression are achieved.